A Review of DNA Enrichment Technologies - Next Generation Sequencing Technologies in Medical Genetics - page 23

Chapter 3
A Review of DNA Enrichment Technologies

3.1 Introduction

Next-generation-sequencing (NGS) technologies, by sequencing hundreds of thou-
sands to millions of DNA templates in parallel, resulted in higher throughput (Gb
scale) and lowered sequencing cost (Mardis 2008 ; Shendure and Ji 2008 ). This has
permitted the defi nition of the entire genome as well as the differences that exist
between them. The ultimate goal is to routinely perform whole-genome sequencing
to allow us to gain a deeper understanding of genetic variation and to defi ne its role
in phenotypic variation and the pathogenesis of complex traits (Mamanova et al.
2010 ). Due to the cost and time limitations, it is not yet feasible to sequence large
numbers of complex genomes. Therefore, a signifi cant effort has focused on the
development of “target enrichment” methods, in which genomic regions are selec-
tively captured from a DNA sample before sequencing (Fig. 3.1 ). This approach is
more time- and cost-effective, and the resulting data are considerably less cumber-
some to analyze, except in the case of exome capture (Chap. 8 ) . Several approaches
to target enrichment have been developed, and the performance parameters vary
from one to another: (1) sensitivity, or the percentage of the target bases that are
represented by one or more sequence reads; (2) specifi city, or the percentage of
sequences that map to the intended targets; (3) uniformity, or the variability in
sequence coverage across target regions; (4) reproducibility, or how closely results
obtained from replicate experiments correlate; (5) cost; (6) ease of use; and (7)
amount of DNA required per experiment, or per megabase of target (Mamanova
et al. 2010 ).
To enrich for regions of interest that range in size from hundreds of kb to the
whole exome, genomic enrichment steps, both traditional and novel, are being
incorporated into overall experimental designs (Voelkerding et al. 2009 ). Traditional
overlapping long-range PCR amplicons (approximately 5-10 kb) can only be used
for up to several hundred kb. More recently, enrichment based on hybridization of
fragmented genomic DNA to oligonucleotide capture probes has been successfully
achieved by several groups (Albert et al. 2007 ; Hodges et al. 2007 ; Okou et al. 2007 ;